Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and polynucleotides encoding the polypeptides, catalytic domains, carbohydrate binding modules and polynucleotides encoding the polypeptides, catalytic domains or carbohydrate binding modules. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or carbohydrate binding modules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/408,470 filed on Dec. 16, 2014, which is a 35 U.S.C. § 371national application of PCT/CN2013/078388 filed on Jun. 28, 2013, whichclaims priority or the benefit under 35 U.S.C. § 119 ofPCT/CN2012/077908 filed on Jun. 29, 2012 and U.S. ProvisionalApplication No. 61/680,898 filed on Aug. 8, 2012, the contents of whichare fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to polypeptides having cellulolyticenhancing activity, catalytic domains, and carbohydrate binding modules,and polynucleotides encoding the polypeptides, catalytic domains, andcarbohydrate binding modules. The invention also relates to nucleic acidconstructs, vectors, and host cells comprising the polynucleotides aswell as methods of producing and using the polypeptides, catalyticdomains, and carbohydrate binding modules.

Description of the Related Art

Cellulose is a polymer of glucose covalently linked by beta-1,4-bonds.Many microorganisms produce enzymes that hydrolyze beta-linked glucans.These enzymes include endoglucanases, cellobiohydrolases, andbeta-glucosidases. Endoglucanases digest the cellulose polymer at randomlocations, opening it to attack by cellobiohydrolases.Cellobiohydrolases sequentially release molecules of cellobiose from theends of the cellulose polymer. Cellobiose is a water-solublebeta-1,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobioseto glucose. Once the cellulose is converted to glucose, the glucose caneasily be fermented by yeast into ethanol.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin.

WO 2005/074647, WO 2008/148131, and WO 2011/035027 disclose isolatedGH61 polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Thielavia terrestris. WO 2005/074656 and WO2010/065830 disclose isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascusaurantiacus. WO 2007/089290 discloses an isolated GH61 polypeptidehaving cellulolytic enhancing activity and the polynucleotide thereoffrom Trichoderma reesei. WO 2009/085935, WO 2009/085859, WO 2009/085864,and WO 2009/085868 disclose isolated GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromMyceliophthora thermophila. WO 2010/138754 discloses an isolated GH61polypeptide having cellulolytic enhancing activity and thepolynucleotide thereof from Aspergillus fumigatus. WO 2011/005867discloses an isolated GH61 polypeptids having cellulolytic enhancingactivity and the polynucleotide thereof from Penicillium pinophilum. WO2011/039319 discloses an isolated GH61 polypeptide having cellulolyticenhancing activity and the polynucleotide thereof from Thermoascus sp.WO 2011/041397 discloses an isolated GH61 polypeptide havingcellulolytic enhancing activity and the polynucleotide thereof fromPenicillium sp. WO 2011/041504 discloses isolated GH61 polypeptideshaving cellulolytic enhancing activity and the polynucleotides thereoffrom Thermoascus crustaceous. WO 2008/151043 discloses methods ofincreasing the activity of a GH61 polypeptide having cellulolyticenhancing activity by adding a soluble activating divalent metal cationto a composition comprising the polypeptide.

There is a need in the art for new enzymes to increase efficiency and toprovide cost-effective enzyme solutions for saccharification ofcellulosic material.

The present invention provides GH61 polypeptides having cellulolyticenhancing activity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingcellulolytic enhancing activity selected from the group consisting of:

(a) a polypeptide having at least 90% sequence identity to the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast very high stringency conditions with the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 3; or the cDNA sequence thereof;or the full-length complement thereof;

(c) a polypeptide encoded by a polynucleotide having at least 90%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or SEQ ID NO: 3; or the cDNA sequences thereof;

(d) a variant of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4comprising a substitution, deletion, and/or insertion at one or more(e.g., several) positions; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to isolated polypeptides comprising acatalytic domain selected from the group consisting of:

(a) a catalytic domain having at least 90% sequence identity to aminoacids 20 to 223 of SEQ ID NO: 4;

(b) a catalytic domain encoded by a polynucleotide that hybridizes underat least very high stringency conditions with nucleotides 58 to 1004 ofSEQ ID NO: 3; the cDNA sequence thereof; or the full-length complementthereof;

(c) a catalytic domain encoded by a polynucleotide having at least 90%sequence identity to nucleotides 58 to 1004 of SEQ ID NO: 3 or the cDNAsequence thereof;

(d) a variant of amino acids 20 to 223 of SEQ ID NO: 4 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the catalytic domain of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to isolated polypeptides comprising acarbohydrate binding module selected from the group consisting of:

(a) a carbohydrate binding module having at least 90% sequence identityto amino acids 255 to 292 of SEQ ID NO: 4;

(b) a carbohydrate binding module encoded by a polynucleotide thathybridizes under at least very high stringency conditions withnucleotides 1098 to 1332 of SEQ ID NO: 3; the cDNA sequence thereof; orthe full-length complement thereof;

(c) a carbohydrate binding module encoded by a polynucleotide having atleast 90% sequence identity to nucleotides 1098 to 1332 of SEQ ID NO: 3or the cDNA sequence thereof;

(d) a variant of amino acids 255 to 292 of SEQ ID NO: 4 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the carbohydrate binding module of (a), (b), (c), or(d) that has binding activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing the polypeptides.

The present invention also relates to processes for degrading acellulosic material, comprising: treating the cellulosic material withan enzyme composition in the presence of a polypeptide havingcellulolytic enhancing activity of the present invention.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme composition in thepresence of a polypeptide having cellulolytic enhancing activity of thepresent invention.

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to17 of SEQ ID NO: 2 or amino acids 1 to 19 of SEQ ID NO: 4, which isoperably linked to a gene encoding a protein, wherein the protein isforeign to the signal peptide; nucleic acid constructs, expressionvectors, and recombinant host cells comprising the polynucleotides; andmethods of producing a protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of pGH61_Mf2197.

FIG. 2 shows a restriction map of pGH61-Mf3680.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Auxiliary Activity 9: The term “Auxiliary Activity 9” or “AA9” means apolypeptide classified as a lytic polysaccharide monooxygenase (Quinlanet al., 2011, Proc. Natl. Acad. Sci. USA 208: 15079-15084; Phillips etal., 2011, ACS Chem. Biol. 6: 1399-1406; Lin et al., 2012, Structure 20:1051-1061; Levasseur et al., 2013, Biotechnology for Biofuels 6: 41).

AA9 polypeptides were formerly classified into the glycoside hydrolaseFamily 61 (GH61) according to Henrissat, 1991, Biochem. J. 280: 309-316,and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, J. Basic Microbiol.42: 55-66. One unit of beta-glucosidase is defined as 1.0 μmole ofp-nitrophenolate anion produced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention,beta-xylosidase activity can be determined using 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20 at pH 5, 40° C. One unit of beta-xylosidaseis defined as 1.0 μmole of p-nitrophenolate anion produced per minute at40° C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside in 100 mM sodiumcitrate containing 0.01% TWEEN® 20.

Carbohydrate binding module: The term “carbohydrate binding module”means the region within a carbohydrate-active enzyme that providescarbohydrate-binding activity (Boraston et al., 2004, Biochem. J. 383:769-781). A majority of known carbohydrate binding modules (CBMs) arecontiguous amino acid sequences with a discrete fold. The carbohydratebinding module (CBM) is typically found either at the N-terminal or atthe C-terminal extremity of an enzyme. Some CBMs are known to havespecificity for cellulose. A specific CBM is a “cellulose bindingdomain”, which means the region of an enzyme that mediates binding ofthe enzyme to amorphous regions of a cellulose substrate. The cellulosebinding domain (CBD) is typically found either at the N-terminal or atthe C-terminal extremity of an enzyme.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing end(cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of thechain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al.,1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity isdetermined according to the procedures described by Lever et al., 1972,Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters,149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187:283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In thepresent invention, the Tomme et al. method can be used to determinecellobiohydrolase activity.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic enzyme activity include:(1) measuring the total cellulolytic enzyme activity, and (2) measuringthe individual cellulolytic enzyme activities (endoglucanases,cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al.,2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzymeactivity can be measured using insoluble substrates, including WhatmanNo 1 filter paper, microcrystalline cellulose, bacterial cellulose,algal cellulose, cotton, pretreated lignocellulose, etc. The most commontotal cellulolytic activity assay is the filter paper assay usingWhatman No 1 filter paper as the substrate. The assay was established bythe International Union of Pure and Applied Chemistry (IUPAC) (Ghose,1987, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in production/release of sugarsduring hydrolysis of a cellulosic material by cellulolytic enzyme(s)under the following conditions: 1-50 mg of cellulolytic enzyme protein/gof cellulose in pretreated corn stover (PCS) (or other pretreatedcellulosic material) for 3-7 days at a suitable temperature such as 40°C.-80° C., e.g., 50° C., 55° C., 60° C., 65° C., or 70° C., and asuitable pH such as 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0, compared to acontrol hydrolysis without addition of cellulolytic enzyme protein.Typical conditions are 1 ml reactions, washed or unwashed PCS, 5%insoluble solids (dry weight), 50 mM sodium acetate pH 5, 1 mM MnSO₄,50° C., 55° C., or 60° C., 72 hours, sugar analysis by AMINEX® HPX-87Hcolumn (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In an embodiment, the cellulosic material is agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, or wood (including forestryresidue).

In another embodiment, the cellulosic material is arundo, bagasse,bamboo, corn cob, corn fiber, corn stover, miscanthus, rice straw,switchgrass, or wheat straw.

In another embodiment, the cellulosic material is aspen, eucalyptus,fir, pine, poplar, spruce, or willow.

In another embodiment, the cellulosic material is algal cellulose,bacterial cellulose, cotton linter, filter paper, microcrystallinecellulose (e.g., AVICEL®), or phosphoric-acid treated cellulose.

In another embodiment, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Endoglucanase: The term “endoglucanase” means a4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3-1,4 glucans suchas cereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat, 1991, Biochem. J.280: 309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.However, the enzymes in this family were recently reclassified as lyticpolysaccharide monooxygenases (Quinlan et al., 2011, Proc. Natl. Acad.Sci. USA 208: 15079-15084; Phillips et al., 2011, ACS Chem. Biol. 6:1399-1406; Lin et al., 2012, Structure 20: 1051-1061) and assigned to anew category called “Auxiliary Activity 9” or “AA9”. The term “GH61” maybe substituted herein with “AA9”.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase (FAE) is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. For purposes of the present invention,feruloyl esterase activity is determined using 0.5 mMp-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. Oneunit of feruloyl esterase equals the amount of enzyme capable ofreleasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has cellulolyticenhancing activity. In one aspect, a fragment contains at least 185amino acid residues, e.g., at least 195 amino acid residues or at least205 amino acid residues of SEQ ID NO: 2. In another aspect, a fragmentcontains at least 230 amino acid residues, e.g., at least 245 amino acidresidues or at least 260 amino acid residues of SEQ ID NO: 4.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom andShoham, Current Opinion In Microbiology, 2003, 6(3): 219-228).Hemicellulases are key components in the degradation of plant biomass.Examples of hemicellulases include, but are not limited to, anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates for theseenzymes, hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families. Some families, with an overall similarfold, can be further grouped into clans, marked alphabetically (e.g.,GH-A). A most informative and updated classification of these and othercarbohydrate active enzymes is available in the Carbohydrate-ActiveEnzymes (CAZy) database. Hemicellulolytic enzyme activities can bemeasured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature such as 40° C.−80° C., e.g., 50°C., 55° C., 60° C., 65° C., or 70° C., and a suitable pH such as 4-9,e.g., 5.0, 5.5, 6.0, 6.5, or 7.0.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 0.2×SSC, 0.2% SDSat 65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 0.2×SSC, 0.2% SDSat 50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 18 to 232 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) thatpredicts amino acids 1 to 17 of SEQ ID NO: 2 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 20 to 292 of SEQID NO: 4 based on the SignalP program that predicts amino acids 1 to 19of SEQ ID NO: 4 are a signal peptide. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellulolytic enhancing activity. In one aspect, the maturepolypeptide coding sequence is nucleotides 52 to 872 of SEQ ID NO: 1 orthe cDNA sequence thereof based on the SignalP program (Nielsen et al.,1997, supra) that predicts nucleotides 1 to 51 of SEQ ID NO: 1 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 58 to 1332 of SEQ ID NO: 3 or the cDNA sequencethereof based on the SignalP program that predicts nucleotides 1 to 57of SEQ ID NO: 3 encode a signal peptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. Cellulolyticenhancing activity can be determined by measuring the increase inreducing sugars or the increase of the total of cellobiose and glucosefrom the hydrolysis of a cellulosic material by cellulolytic enzymeunder the following conditions: 1-50 mg of total protein/g of cellulosein pretreated corn stover (PCS), wherein total protein is comprised of50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of aGH61 polypeptide having cellulolytic enhancing activity for 1-7 days ata suitable temperature, such as 40° C.-80° C., e.g., 50° C., 55° C., 60°C., 65° C., or 70° C., and a suitable pH, such as 4-9, e.g., 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5, compared to a control hydrolysiswith equal total protein loading without cellulolytic enhancing activity(1-50 mg of cellulolytic protein/g of cellulose in PCS). In a preferredaspect, a mixture of CELLUCLAST® 1.5L (Novozymes A/S, Bagsværd, Denmark)in the presence of 2-3% of total protein weight Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae accordingto WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus oryzae asdescribed in WO 02/095014) of cellulase protein loading is used as thesource of the cellulolytic activity.

GH61 polypeptide enhancing activity can also be determined by incubatingthe GH61 polypeptide with 0.5% phosphoric acid swollen cellulose (PASO),100 mM sodium acetate pH 5, 1 mM MnSO₄, 0.1% gallic acid, 0.025 mg/ml ofAspergillus fumigatus beta-glucosidase, and 0.01% TRITON® X-100(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 24-96 hoursat 40° C. followed by determination of the glucose released from thePASO.

GH61 polypeptide enhancing activity can also be determined according toWO 2013/028928 for high temperature compositions. GH61 polypeptideenhancing activity can also be determined according to Examples 8-11herein.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

The polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, and at least 100% of the cellulolytic enhancingactivity of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

Pretreated corn stover: The term “Pretreated Corn Stover” or “PCS” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, neutral pretreatment,or any pretreatment known in the art.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 3.0.0, 5.0.0 or later. The parametersused are a gap open penalty of 10, a gap extension penalty of 0.5, andthe EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. Theoutput of Needle labeled “longest identity” (obtained using the −nobriefoption) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 3.0.0, 5.0.0 or later. The parameters used are a gap openpenalty of 10, q gap extension penalty of 0.5, and the EDNAFULL (EMBOSSversion of NCBI NUC4.4) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows: (IdenticalDeoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps inAlignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having cellulolytic enhancing activity. In one aspect, asubsequence contains at least 555 nucleotides, e.g., at least 585nucleotides or at least 615 nucleotides of SEQ ID NO: 1. In anotheraspect, a subsequence contains at least 690 nucleotides, e.g., at least735 nucleotides or at least 780 nucleotides of SEQ ID NO: 3.

Variant: The term “variant” means a polypeptide having cellulolyticenhancing activity comprising an alteration, i.e., a substitution,insertion, and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 45° C.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the processes of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, 2006, Journal of the Science of Food and Agriculture 86(11):1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19): 4597-4601;Herrmann et al., 1997, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. A common total xylanolytic activity assay is based onproduction of reducing sugars from polymeric 4-O-methyl glucuronoxylanas described in Bailey, Biely, Poutanen, 1992, Interlaboratory testingof methods for assay of xylanase activity, Journal of Biotechnology23(3): 257-270. Xylanase activity can also be determined with 0.2%AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodiumphosphate pH 6 at 37° C. One unit of xylanase activity is defined as 1.0μmole of azurine produced per minute at 37° C., pH 6 from 0.2%AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

Xylan degrading activity can be determined by measuring the increase inhydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo.,USA) by xylan-degrading enzyme(s) under the following typicalconditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg ofxylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50° C.,24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH)assay as described by Lever, 1972, Anal. Biochem. 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37° C. One unit ofxylanase activity is defined as 1.0 μmole of azurine produced per minuteat 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mMsodium phosphate pH 6.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having CellulolyticEnhancing Activity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 orSEQ ID NO: 4 of at least 90%, e.g., at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%, which have cellulolytic enhancing activity.In one aspect, the polypeptides differ by up to 10 amino acids, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO:2.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or an allelicvariant thereof; or is a fragment thereof having cellulolytic enhancingactivity. In another aspect, the polypeptide comprises or consists ofthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4. In anotheraspect, the polypeptide comprises or consists of amino acids 18 to 232of SEQ ID NO: 2 or amino acids 20 to 292 of SEQ ID NO: 4.

In another embodiment, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity encoded bypolynucleotides that hybridize under very low stringency conditions, lowstringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequence of SEQ ID NO: 1 orSEQ ID NO: 3 or (iii) the full-length complement of (i) or (ii)(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, New York).

The polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 3, or a subsequencethereof, as well as the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, themature polypeptide thereof, or a fragment thereof, may be used to designnucleic acid probes to identify and clone DNA encoding polypeptideshaving cellulolytic enhancing activity from strains of different generaor species according to methods well known in the art. In particular,such probes can be used for hybridization with the genomic DNA or cDNAof a cell of interest, following standard Southern blotting procedures,in order to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 15, e.g., at least 25, at least 35, or at least 70nucleotides in length. Preferably, the nucleic acid probe is at least100 nucleotides in length, e.g., at least 200 nucleotides, at least 300nucleotides, at least 400 nucleotides, at least 500 nucleotides, atleast 600 nucleotides, at least 700 nucleotides, at least 800nucleotides, or at least 900 nucleotides in length. Both DNA and RNAprobes can be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having cellulolytic enhancing activity. Genomic orother DNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that hybridizes with SEQ ID NO: 1 orSEQ ID NO: 3, the mature polypeptide coding sequence thereof, or asubsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1 or SEQ ID NO: 3; (ii) the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 3; (iii) the cDNA sequence of SEQID NO: 1 or SEQ ID NO: 3; (iv) the full-length complement thereof; or(v) a subsequence thereof; under very low to very high stringencyconditions. Molecules to which the nucleic acid probe hybridizes underthese conditions can be detected using, for example, X-ray film or anyother detection means known in the art.

In one aspect, the nucleic acid probe is a polynucleotide that encodesthe polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4; the mature polypeptidethereof; or a fragment thereof. In another aspect, the nucleic acidprobe is SEQ ID NO: 1 or SEQ ID NO: 3; the mature polypeptide codingsequence thereof 3; or the cDNA sequence thereof. In another aspect, thenucleic acid probe is the polynucleotide contained in Corynascusthermophilus CBS 174.70, wherein the polynucleotide encodes apolypeptide having cellulolytic enhancing activity. In another aspect,the nucleic acid probe is the mature polypeptide coding region containedin Corynascus thermophilus CBS 174.70.

In another embodiment, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity encoded bypolynucleotides having a sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 or SEQ ID NO: 3 of at least 90%, e.g.,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In one aspect, the number of amino acid substitutions,deletions and/or insertions introduced into the mature polypeptide ofSEQ ID NO: 2 or SEQ ID NO: 4 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, or 10. The amino acid changes may be of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R.L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for cellulolytic enhancing activity to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity ofessential amino acids can also be inferred from an alignment with arelated polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Cellulolytic Enhancing Activity

A polypeptide having cellulolytic enhancing activity of the presentinvention may be obtained from microorganisms of any genus. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby a polynucleotide is produced by the source or by a strain in whichthe polynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a fungal polypeptide. In one aspect, thepolypeptide is a Corynascus polypeptide. In another aspect, thepolypeptide is a Corynascus thermophilus polypeptide. In another aspect,the polypeptide is a Corynascus thermophilus CBS 174.70 polypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Catalytic Domains

In one embodiment, the present invention relates to catalytic domainshaving a sequence identity to amino acids 20 to 223 of SEQ ID NO: 4 ofat least 90%, e.g., at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%. In one aspect, the catalytic domains comprise amino acidsequences that differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, from amino acids 20 to 223 of SEQ ID NO: 4.

The catalytic domain preferably comprises or consists of amino acids 20to 223 of SEQ ID NO: 4, or an allelic variant thereof; or is a fragmentthereof having cellulolytic enhancing activity.

In another embodiment, the present invention relates to catalyticdomains encoded by polynucleotides that hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions (as defined above) withnucleotides 58 to 1004 of SEQ ID NO: 3; the cDNA sequence thereof; orthe full-length complement thereof (Sambrook et al., 1989, supra).

In another embodiment, the present invention relates to catalyticdomains encoded by polynucleotides having a sequence identity tonucleotides 58 to 1004 of SEQ ID NO: 3 or the cDNA sequence thereof ofat least 90%, e.g., at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%.

The polynucleotide encoding the catalytic domain preferably comprises orconsists of nucleotides 58 to 1004 of SEQ ID NO: 3 or the cDNA sequencethereof, or is the sequence contained in Corynascus thermophilus CBS174.70.

In another embodiment, the present invention relates to catalytic domainvariants of amino acids 20 to 223 of SEQ ID NO: 4 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In one aspect, the number of amino acid substitutions,deletions and/or insertions introduced into the sequence of amino acids20 to 223 of SEQ ID NO: 4 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or10.

Carbohydrate Binding Modules

In one embodiment, the present invention relates to carbohydrate bindingmodules having a sequence identity to amino acids 255 to 292 of SEQ IDNO: 4 of at least 90%, e.g., at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In one aspect, the carbohydrate binding modulescomprise amino acid sequences that differ by up to 10 amino acids, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 255 to 292 of SEQ IDNO: 4.

The carbohydrate binding module preferably comprises or consists ofamino acids 255 to 292 of SEQ ID NO: 4, or an allelic variant thereof;or is a fragment thereof having cellulose binding activity.

In another embodiment, the present invention relates to carbohydratebinding modules encoded by polynucleotides that hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions (as defined above) withnucleotides 1098 to 1332 of SEQ ID NO: 3; the cDNA sequence thereof, orthe full-length complement thereof (Sambrook et al., 1989, supra).

In another embodiment, the present invention relates to carbohydratebinding modules encoded by polynucleotides having a sequence identity tonucleotides 1098 to 1332 of SEQ ID NO: 3 or the cDNA sequence thereof ofat least 90%, e.g., at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%.

The polynucleotide encoding the carbohydrate binding module preferablycomprises or consists of nucleotides 1098 to 1332 of SEQ ID NO: 3 or thecDNA sequence thereof, or is the sequence contained in Corynascusthermophilus CBS 174.70.

In another embodiment, the present invention relates to carbohydratebinding module variants of amino acids 255 to 292 of SEQ ID NO: 4comprising a substitution, deletion, and/or insertion at one or more(e.g., several) positions. In one aspect, the number of amino acidsubstitutions, deletions and/or insertions introduced into the sequenceof amino acids 255 to 292 of SEQ ID NO: 4 is up to 10, e.g., 1, 2, 3, 4,5, 6, 8, 9, or 10.

A catalytic domain operably linked to the carbohydrate binding modulemay be from a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, xylanase, or beta-xylosidase. The polynucleotideencoding the catalytic domain may be obtained from any prokaryotic,eukaryotic, or other source.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide, a catalytic domain, or carbohydrate binding module of thepresent invention, as described herein.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well-known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Corynascus, or a relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO:3, or the cDNA sequence thereof, by introduction of nucleotidesubstitutions that do not result in a change in the amino acid sequenceof the polypeptide, but which correspond to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions that may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxam ide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive or Gram negativebacterium. Gram positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect lmmun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp. 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and optionally (b) recovering thepolypeptide. In one aspect, the cell is a Corynascus cell. In anotheraspect, the cell is a Corynascus thermophilus cell. In another aspect,the cell is Corynascus thermophilus CBS 174.70.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a whole fermentation broth comprising apolypeptide of the present invention is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideso as to express and produce a polypeptide or domain (e.g., catalyticdomain or carbohydrate binding module) of the present invention inrecoverable quantities. The polypeptide or domain may be recovered fromthe plant or plant part. Alternatively, the plant or plant partcontaining the polypeptide or domain may be used as such for improvingthe quality of a food or feed, e.g., improving nutritional value,palatability, and rheological properties, or to destroy an antinutritivefactor

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art. Inshort, the plant or plant cell is constructed by incorporating one ormore expression constructs encoding the polypeptide or domain into theplant host genome or chloroplast genome and propagating the resultingmodified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain operablylinked with appropriate regulatory sequences required for expression ofthe polynucleotide in the plant or plant part of choice. Furthermore,the expression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainis desired to be expressed (Sticklen, 2008, Nature Rev. 9: 433-443). Forinstance, the expression of the gene encoding a polypeptide or domainmay be constitutive or inducible, or may be developmental, stage ortissue specific, and the gene product may be targeted to a specifictissue or plant part such as seeds or leaves. Regulatory sequences are,for example, described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain in the plant. For instance, thepromoter enhancer element may be an intron that is placed between thepromoter and the polynucleotide encoding a polypeptide or domain. Forinstance, Xu et al., 1993, supra, disclose the use of the first intronof the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain canbe introduced into a particular plant variety by crossing, without theneed for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention. Crossing results in the introduction of a transgene into aplant line by cross pollinating a starting line with a donor plant line.Non-limiting examples of such steps are described in U.S. Pat. No.7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and (b) recovering the polypeptide or domain.

Removal or Reduction of Cellulolytic Enhancing Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions. In oneaspect, the parent cell is a Corynascus thermophilus cell.

The mutant cell may be constructed by reducing or eliminating expressionof the polynucleotide using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. In a preferredaspect, the polynucleotide is inactivated. The polynucleotide to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required forexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thepolynucleotide. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed bysubjecting the parent cell to mutagenesis and selecting for mutant cellsin which expression of the polynucleotide has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the polynucleotide may also beaccomplished by insertion, substitution, or deletion of one or morenucleotides in the gene or a regulatory element required fortranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing thepolynucleotide to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of apolynucleotide is based on techniques of gene replacement, genedeletion, or gene disruption. For example, in the gene disruptionmethod, a nucleic acid sequence corresponding to the endogenouspolynucleotide is mutagenized in vitro to produce a defective nucleicacid sequence that is then transformed into the parent cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous polynucleotide. It may be desirablethat the defective polynucleotide also encodes a marker that may be usedfor selection of transformants in which the polynucleotide has beenmodified or destroyed. In an aspect, the polynucleotide is disruptedwith a selectable marker such as those described herein.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having cellulolytic enhancing activity in acell, comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA forinhibiting transcription. In another preferred aspect, the dsRNA ismicro RNA for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 3 for inhibiting expression ofthe polypeptide in a cell. While the present invention is not limited byany particular mechanism of action, the dsRNA can enter a cell and causethe degradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing a dsRNAi of the present invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art; see, for example, U.S. Pat. Nos.6,489,127; 6,506,559; 6,511,824; and 6,515,109.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a polynucleotide encoding thepolypeptide or a control sequence thereof or a silenced gene encodingthe polypeptide, which results in the mutant cell producing less of thepolypeptide or no polypeptide compared to the parent cell.

The polypeptide-deficient mutant cells are particularly useful as hostcells for expression of native and heterologous polypeptides. Therefore,the present invention further relates to methods of producing a nativeor heterologous polypeptide, comprising (a) cultivating the mutant cellunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. The term “heterologous polypeptides” meanspolypeptides that are not native to the host cell, e.g., a variant of anative protein. The host cell may comprise more than one copy of apolynucleotide encoding the native or heterologous polypeptide. Forexample, the parent cell may be a producer of a cellulolytic mixture ofenzymes, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, andGH61 polypeptides.Optimal performance of the cellulolytic enzyme mixturecan depend on several factors including, but not limited to, the mixtureof cellulase enzymes, the cellulosic material, the concentration ofcellulosic material, the pretreatment of the cellulosic material,temperature, time, and pH. One or more of the cellulolytic enzymes maynot perform optimally at a high temperature. In order to improve theperformance of the cellulolytic enzyme mixture, it may be desirable toreplace the polypeptide having cellulolytic enhancing activity with adifferent polypeptide having cellulolytic enhancing activity. Oneskilled in the art would accomplish this replacement by inactivating thegene encoding the polypeptide having cellulolytic enhancing activity ofthe present invention in the parent cell and replacing it with adifferent gene encoding another polypeptide having cellulolyticenhancing activity with an improved property, e.g., thermostability orthermal activity.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallycellulolytic enhancing activity-free product are of particular interestin the production of eukaryotic polypeptides, in particular fungalproteins such as enzymes. The cellulolytic enhancing activity-deficientcells may also be used to express heterologous proteins ofpharmaceutical interest such as hormones, growth factors, receptors, andthe like. The term “eukaryotic polypeptides” includes not only nativepolypeptides, but also those polypeptides, e.g., enzymes, which havebeen modified by amino acid substitutions, deletions or additions, orother such modifications to enhance activity, thermostability, pHtolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from cellulolytic enhancing activity that is producedby a method of the present invention.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.The fermentation broth product further comprises additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. In some embodiments, the composition is a cell-killed wholebroth containing organic acid(s), killed cells and/or cell debris, andculture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compostions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The fermentation broth formulations or cell compositions may furthercomprise multiple enzymatic activities, such as one or more (e.g.,several) enzymes selected from the group consisting of a cellulase, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Thefermentation broth formulations or cell compositions may also compriseone or more (e.g., several) enzymes selected from the group consistingof a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or atransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Enzyme Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecellulolytic enhancing activity of the composition has been increased,e.g., with an enrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin. The compositions may also comprise one ormore (e.g., several) enzymes selected from the group consisting of ahydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or atransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

The present invention is also directed to the following processes forusing the polypeptides having cellulolytic enhancing activity, orcompositions thereof.

The present invention also relates to processes for degrading acellulosic material, comprising: treating the cellulosic material withan enzyme composition in the presence of a polypeptide havingcellulolytic enhancing activity of the present invention. In one aspect,the processes further comprise recovering the degraded cellulosicmaterial. Soluble products of degradation or conversion of thecellulosic material can be separated from insoluble cellulosic materialusing a method known in the art such as, for example, centrifugation,filtration, or gravity settling.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme composition in thepresence of a polypeptide having cellulolytic enhancing activity of thepresent invention. In one aspect, the fermenting of the cellulosicmaterial produces a fermentation product. In another aspect, theprocesses further comprise recovering the fermentation product from thefermentation.

The processes of the present invention can be used to saccharify thecellulosic material to fermentable sugars and to convert the fermentablesugars to many useful fermentation products, e.g., fuel (ethanol,n-butanol, isobutanol, biodiesel, jet fuel) and/or platform chemicals(e.g., acids, alcohols, ketones, gases, oils, and the like). Theproduction of a desired fermentation product from the cellulosicmaterial typically involves pretreatment, enzymatic hydrolysis(saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using methods conventional in the art.Moreover, the processes of the present invention can be implementedusing any conventional biomass processing apparatus configured tooperate in accordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehanand Himmel, 1999, Biotechnol. Prog. 15: 817-827). HHF involves aseparate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd et al., 2002,Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein thatany method known in the art comprising pretreatment, enzymatichydrolysis (saccharification), fermentation, or a combination thereof,can be used in the practicing the processes of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (deCastilhos Corazza et al., 2003, Acta Scientiarum. Technology 25: 33-38;Gusakov and Sinitsyn, 1985, Enz. Microb. Technol. 7: 346-352), anattrition reactor (Ryu and Lee, 1983, Biotechnol. Bioeng. 25: 53-65).Additional reactor types include fluidized bed, upflow blanket,immobilized, and extruder type reactors for hydrolysis and/orfermentation.

Pretreatment.

In practicing the processes of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of the cellulosic material (Chandra et al., 2007, Adv.Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Adv.Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009,Bioresource Technology 100: 10-18; Mosier et al., 2005, BioresourceTechnology 96: 673-686; Taherzadeh and Karimi, 2008, Int. J. Mol. Sci.9: 1621-1651; Yang and Wyman, 2008, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C.,where the optimal temperature range depends on optional addition of achemical catalyst. Residence time for the steam pretreatment ispreferably 1-60 minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes,or 4-10 minutes, where the optimal residence time depends on thetemperature and optional addition of a chemical catalyst. Steampretreatment allows for relatively high solids loadings, so that thecellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 2002/0164730). During steam pretreatment, hemicellulose acetylgroups are cleaved and the resulting acid autocatalyzes partialhydrolysis of the hemicellulose to monosaccharides and oligosaccharides.Lignin is removed to only a limited extent.

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexpansion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A chemical catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) issometimes added prior to steam pretreatment, which decreases the timeand temperature, increases the recovery, and improves enzymatichydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol.129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol.113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39:756-762). In dilute acid pretreatment, the cellulosic material is mixedwith dilute acid, typically H₂SO₄, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technology 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze expansion (AFEX) pretreatment.

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technology 96: 1959-1966; Mosieret al., 2005, Bioresource Technology 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technology 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber expansion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technology 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. Biotechnol. 105-108: 69-85, and Mosier etal., 2005, Bioresource Technology 96: 673-686, and U.S. PublishedApplication 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or0.1 to 2 wt % acid. The acid is contacted with the cellulosic materialand held at a temperature in the range of preferably 140-200° C., e.g.,165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt %or 30-60 wt %, such as around 40 wt %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperature in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hem icellulose, and/or lignin from the cellulosicmaterial. Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Adv. Appl. Microbiol.39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass:a review, in Enzymatic Conversion of Biomass for Fuels Production,Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS SymposiumSeries 566, American Chemical Society, Washington, D.C., chapter 15;Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanolproduction from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996, Enz.Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Adv.Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose and/orhemicellulose to fermentable sugars, such as glucose, cellobiose,xylose, xylulose, arabinose, mannose, galactose, and/or solubleoligosaccharides. The hydrolysis is performed enzymatically by an enzymecomposition in the presence of a polypeptide having cellulolyticenhancing activity of the present invention. The enzymes of thecompositions can be added simultaneously or sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzymes(s), i.e., optimalfor the enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material is fed gradually to,for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 8, e.g., about 3.5 to about 7,about 4 to about 6, or about 4.5 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt. %, e.g., about 10 toabout 40 wt. % or about 20 to about 30 wt. %.

The enzyme compositions can comprise any protein useful in degrading thecellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Inanother aspect, the cellulase is preferably one or more (e.g., several)enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase. In another aspect, thehemicellulase is preferably one or more (e.g., several) enzymes selectedfrom the group consisting of an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase and a polypeptidehaving cellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a cellobiohydrolase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a beta-glucosidase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises an endoglucanase and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase and a beta-glucosidase. Inanother aspect, the enzyme composition comprises a beta-glucosidase anda cellobiohydrolase. In another aspect, the enzyme composition comprisesa beta-glucosidase and a cellobiohydrolase I, a cellobiohydrolase II, ora combination of a cellobiohydrolase I and a cellobiohydrolase II. Inanother aspect, the enzyme composition comprises an endoglucanase, apolypeptide having cellulolytic enhancing activity, and acellobiohydrolase. In another aspect, the enzyme composition comprisesan endoglucanase, a polypeptide having cellulolytic enhancing activity,and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase, a beta-glucosidase, and apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises a beta-glucosidase, a polypeptidehaving cellulolytic enhancing activity, and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises a beta-glucosidase, apolypeptide having cellulolytic enhancing activity, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase, a beta-glucosidase, and acellobiohydrolase. In another aspect, the enzyme composition comprisesan endoglucanase, a beta-glucosidase, and a cellobiohydrolase I, acellobiohydrolase II, or a combination of a cellobiohydrolase I and acellobiohydrolase II. In another aspect, the enzyme compositioncomprises an endoglucanase, a cellobiohydrolase, a beta-glucosidase, anda polypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase, a beta-glucosidase, apolypeptide having cellulolytic enhancing activity, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In anembodiment, the xylanase is a Family 10 xylanase. In another embodiment,the xylanase is a Family 11 xylanase. In another aspect, the enzymecomposition comprises a xylosidase (e.g., beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a laccase. In another aspect,the enzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin.

In the processes of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may benative proteins, recombinant proteins, or a combination of nativeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. It is understood herein that therecombinant proteins may be heterologous (e.g., foreign) and/or nativeto the host cell. One or more (e.g., several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the processes of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes and a polypeptide having cellulolyticenhancing activity depend on several factors including, but not limitedto, the mixture of cellulolytic enzymes and/or hemicellulolytic enzymes,the cellulosic material, the concentration of cellulosic material, thepretreatment(s) of the cellulosic material, temperature, time, pH, andinclusion of a fermenting organism (e.g., for SimultaneousSaccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to the cellulosic material is about 0.01to about 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg,about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 toabout 1.25 mg, or about 0.25 to about 1.0 mg per g of the cellulosicmaterial.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to cellulolytic or hemicellulolyticenzyme is about 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0 g,about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 toabout 0.5 g, about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g perg of cellulolytic or hemicellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material (collectively hereinafter“polypeptides having enzyme activity”) can be derived or obtained fromany suitable origin, including, archaeal, bacterial, fungal, yeast,plant, or animal origin. The term “obtained” also means herein that theenzyme may have been produced recombinantly in a host organism employingmethods described herein, wherein the recombinantly produced enzyme iseither native or foreign to the host organism or has a modified aminoacid sequence, e.g., having one or more (e.g., several) amino acids thatare deleted, inserted and/or substituted, i.e., a recombinantly producedenzyme that is a mutant and/or a fragment of a native amino acidsequence or an enzyme produced by nucleic acid shuffling processes knownin the art. Encompassed within the meaning of a native enzyme arenatural variants and within the meaning of a foreign enzyme are variantsobtained by, e.g., site-directed mutagenesis or shuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a Gram positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus,Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacilluspolypeptide having enzyme activity, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide having enzyme activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having enzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In one aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having enzyme activity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host can be a heterologous host (enzyme is foreign tohost), but the host may under certain conditions also be a homologoushost (enzyme is native to host). Monocomponent cellulolytic proteins mayalso be prepared by purifying such a protein from a fermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S),CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), SPEZYME™ CP(Genencor Int.), ACCELERASE™ TRIO (DuPont), FILTRASE® NL (DSM);METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Röhm GmbH), or ALTERNAFUEL®CMAX3™ (Dyadic International, Inc.). The cellulolytic enzyme preparationis added in an amount effective from about 0.001 to about 5.0 wt. % ofsolids, e.g., about 0.025 to about 4.0 wt. % of solids or about 0.005 toabout 2.0 wt. % of solids.

Examples of bacterial endoglucanases that can be used in the processesof the present invention, include, but are not limited to, Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655; WO 00/70031; WO05/093050), Erwinia carotovara endoglucanase (Saarilahti et al., 1990,Gene 90: 9-14), Thermobifida fusca endoglucanase III (WO 05/093050), andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GenBank:M15665), Trichoderma reeseiendoglucanase II (Saloheimo, et al., 1988, Gene 63:11-22), Trichodermareesei Cel5A endoglucanase II (GenBank:M19373), Trichoderma reeseiendoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64:555-563, GenBank:AB003694), Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228,GenBank:Z33381), Aspergillus aculeatus endoglucanase (Ooi et al., 1990,Nucleic Acids Research 18: 5884), Aspergillus kawachii endoglucanase(Sakamoto et al., 1995, Current Genetics 27: 435-439), Erwiniacarotovara endoglucanase (Saarilahti et al., 1990, Gene 90: 9-14),Fusarium oxysporum endoglucanase (GenBank:L29381), Humicola grisea var.thermoidea endoglucanase (GenBank:AB003107), Melanocarpus albomycesendoglucanase (GenBank:MAL515703), Neurospora crassa endoglucanase(GenBank:XM_324477), Humicola insolens endoglucanase V, Myceliophthorathermophila CBS 117.65 endoglucanase, basidiomycete CBS 495.95endoglucanase, basidiomycete CBS 494.95 endoglucanase, Thielaviaterrestris NRRL 8126 CEL6B endoglucanase, Thielavia terrestris NRRL 8126CEL6C endoglucanase, Thielavia terrestris NRRL 8126 CEL7C endoglucanase,Thielavia terrestris NRRL 8126 CEL7E endoglucanase, Thielavia terrestrisNRRL 8126 CEL7F endoglucanase, Cladorrhinum foecundissimum ATCC 62373CEL7A endoglucanase, and Trichoderma reesei strain No. VTT-D-80133endoglucanase (GenBank:M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Penicillium occitanis cellobiohydrolase I (GenBank:AY690482),Talaromyces emersonii cellobiohydrolase I (GenBank:AF439936), Thielaviahyrcanie cellobiohydrolase II (WO 2010/141325), Thielavia terrestriscellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 02/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BGfusion protein (WO 2008/057637) or an Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637.

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat, 1991, Biochem. J. 280: 309-316,and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO2008/008070, WO 2008/008793, U.S. Pat. Nos. 5,457,046, 5,648,263, and5,686,593.

In one aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a soluble activating divalent metalcation according to WO 2008/151043, e.g., manganese or copper.

In another aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a dioxy compound, a bicyliccompound, a heterocyclic compound, a nitrogen-containing compound, aquinone compound, a sulfur-containing compound, or a liquor obtainedfrom a pretreated cellulosic material such as pretreated corn stover (WO2012/021394, WO 2012/021395, WO 2012/021396, WO 2012/021399, WO2012/021400, WO 2012/021401, WO 2012/021408, and WO 2012/021410)).

The dioxy compound may include any suitable compound containing two ormore oxygen atoms. In some aspects, the dioxy compounds contain asubstituted aryl moiety as described herein. The dioxy compounds maycomprise one or more (e.g., several) hydroxyl and/or hydroxylderivatives, but also include substituted aryl moieties lacking hydroxyland hydroxyl derivatives. Non-limiting examples of the dioxy compoundsinclude pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoicacid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethylgallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol;(croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol;3-ethyoxy-1,2-propanediol; 2,4,4′-trihydroxybenzophenone;cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione;dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate;4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or asalt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ringsystem as described herein. The compounds may comprise one or more(e.g., several) additional rings, and are not limited to a specificnumber of rings unless otherwise stated. In one aspect, the bicycliccompound is a flavonoid. In another aspect, the bicyclic compound is anoptionally substituted isoflavonoid. In another aspect, the bicycliccompound is an optionally substituted flavylium ion, such as anoptionally substituted anthocyanidin or optionally substitutedanthocyanin, or derivative thereof. Non-limiting examples of thebicyclic compounds include epicatechin; quercetin; myricetin; taxifolin;kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin;cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as anoptionally substituted aromatic or non-aromatic ring comprising aheteroatom, as described herein. In one aspect, the heterocyclic is acompound comprising an optionally substituted heterocycloalkyl moiety oran optionally substituted heteroaryl moiety. In another aspect, theoptionally substituted heterocycloalkyl moiety or optionally substitutedheteroaryl moiety is an optionally substituted 5-memberedheterocycloalkyl or an optionally substituted 5-membered heteroarylmoiety. In another aspect, the optionally substituted heterocycloalkylor optionally substituted heteroaryl moiety is an optionally substitutedmoiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl,dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl,pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl,piperidinyl, and oxepinyl. In another aspect, the optionally substitutedheterocycloalkyl moiety or optionally substituted heteroaryl moiety isan optionally substituted furanyl. Non-limiting examples of theheterocyclic compounds include(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone;ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconicacid δ-lactone; 4-hydroxycoumarin; dihydrobenzofuran;5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;5,6-dihydro-2H-pyran-2-one; and5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvatethereof.

The nitrogen-containing compound may be any suitable compound with oneor more nitrogen atoms. In one aspect, the nitrogen-containing compoundcomprises an amine, imine, hydroxylamine, or nitroxide moiety.Non-limiting examples of the nitrogen-containing compounds includeacetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol;1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy;5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; andmaleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinonemoiety as described herein. Non-limiting examples of the quinonecompounds include 1,4-benzoquinone; 1,4-naphthoquinone;2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone orcoenzyme Q₀; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione oradrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinolinequinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprisingone or more sulfur atoms. In one aspect, the sulfur-containing comprisesa moiety selected from thionyl, thioether, sulfinyl, sulfonyl,sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limitingexamples of the sulfur-containing compounds include ethanethiol;2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid;benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione;cystine; or a salt or solvate thereof.

In one aspect, an effective amount of such a compound described above tocellulosic material is added at a molar ratio to glucosyl units ofcellulose of about 10⁻⁶ to about 10, e.g., about 10⁻⁶ to about 7.5,about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5, about 10⁻⁶ to about 1,about 10⁻⁵ to about 1, about 10⁻⁵ to about 10⁻¹, about 10⁻⁴ to about10⁻¹, about 10⁻³ to about 10⁻¹, or about 10⁻³ to about 10⁻². In anotheraspect, an effective amount of such a compound described above is about0.1 μM to about 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μMto about 0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M,about 5 μM to about 50 mM, about 10 μM to about 25 mM, about 50 μM toabout 25 mM, about 10 μM to about 10 mM, about 5 μM to about 5 mM, orabout 0.1 mM to about 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described in WO2012/021401, and the soluble contents thereof. A liquor for cellulolyticenhancement of a GH61 polypeptide can be produced by treating alignocellulose or hemicellulose material (or feedstock) by applying heatand/or pressure, optionally in the presence of a catalyst, e.g., acid,optionally in the presence of an organic solvent, and optionally incombination with physical disruption of the material, and thenseparating the solution from the residual solids. Such conditionsdetermine the degree of cellulolytic enhancement obtainable through thecombination of liquor and a GH61 polypeptide during hydrolysis of acellulosic substrate by a cellulolytic enzyme preparation. The liquorcan be separated from the treated material using a method standard inthe art, such as filtration, sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about 1g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC®HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (NovozymesA/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), and DEPOL™762P (Biocatalysts Limit, Wales, UK), ALTERNA FUEL 100P (Dyadic), andALTERNA FUEL 200P (Dyadic).

Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Talaromyces lanuginosus GH11 (WO 2012/130965),Talaromyces thermophilus GH11 (WO 2012/13095), Thielavia terrestris NRRL8126 (WO 2009/079210), and Trichophaea saccata GH10 (WO 2011/057083).

Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt:Q7SOW4), Trichoderma reesei(UniProtKB/TrEMBL:Q92458), Talaromyces emersonii (SwissProt: Q8X212),and Talaromyces thermophilus GH11 (WO 2012/13095).

Examples of acetylxylan esterases useful in the processes of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprotaccession number Q2GWX4), Chaetomium gracile (GeneSeqP accession numberAAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocreajecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880),Neurospora crassa (UniProt accession number q7s259), Phaeosphaerianodorum (Uniprot accession number Q0UHJ1), and Thielavia terrestris NRRL8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in theprocesses of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt:A1D9T4), Neurospora crassa(UniProt:Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), andThielavia terrestris (WO 2010/053838 and WO 2010/065448).

Examples of arabinofuranosidases useful in the processes of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP:AAR94170), Humicola insolens DSM 1800 (WO2006/114094 and WO 2009/073383), and M. giganteus (WO 2006/114094).

Examples of alpha-glucuronidases useful in the processes of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt:alcc12), Aspergillus fumigatus(SwissProt:Q4VWV45), Aspergillus niger (UniProt:Q96WX9), Aspergillusterreus (SwissProt:Q0CJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt:Q8X211), and Trichoderma reesei (UniProt:Q99024).

The polypeptides having enzyme activity used in the processes of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, C A, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, N Y, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (e.g., several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on economics, i.e., costs per equivalent sugarpotential, and recalcitrance to enzymatic conversion.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Yeast includestrains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candidasonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Xylose fermenting yeast include strains of Candida, preferably C.sheatae or C. sonorensis; and strains of Pichia, e.g., P. stipitis, suchas P. stipitis CBS 5773. Pentose fermenting yeast include strains ofPachysolen, preferably P. tannophilus. Organisms not capable offermenting pentose sugars, such as xylose and arabinose, may begenetically modified to do so by methods known in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In an aspect, the fermenting microorganism has been genetically modifiedto provide the ability to ferment pentose sugars, such as xyloseutilizing, arabinose utilizing, and xylose and arabinose co-utilizingmicroorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Appl.Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Appl. Environ.Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Appl. Microbiol.Biotechnol. 38: 776-783; Walfridsson et al., 1995, Appl. Environ.Microbiol. 61: 4184-4190; Kuyper et al., 2004, FEMS Yeast Research 4:655-664; Beall et al., 1991, Biotech. Bioeng. 38: 296-303; Ingram etal., 1998, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Science267: 240-243; Deanda et al., 1996, Appl. Environ. Microbiol. 62:4465-4470; WO 03/062430).

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide. The fermentation product can also beprotein as a high value product.

In one aspect, the fermentation product is an alcohol. It will beunderstood that the term “alcohol” encompasses a substance that containsone or more hydroxyl moieties. The alcohol can be, but is not limitedto, n-butanol, isobutanol, ethanol, methanol, arabinitol, butanediol,ethylene glycol, glycerin, glycerol, 1,3-propanediol, sorbitol, xylitol.See, for example, Gong et al., 1999, Ethanol production from renewableresources, in Advances in Biochemical Engineering/Biotechnology,Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65:207-241; Silveira and Jonas, 2002, Appl. Microbiol. Biotechnol. 59:400-408; Nigam and Singh, 1995, Process Biochemistry 30(2): 117-124;Ezeji et al., 2003, World Journal of Microbiology and Biotechnology19(6): 595-603.

In another aspect, the fermentation product is an alkane. The alkane maybe an unbranched or a branched alkane. The alkane can be, but is notlimited to, pentane, hexane, heptane, octane, nonane, decane, undecane,or dodecane.

In another aspect, the fermentation product is a cycloalkane. Thecycloalkane can be, but is not limited to, cyclopentane, cyclohexane,cycloheptane, or cyclooctane.

In another aspect, the fermentation product is an alkene. The alkene maybe an unbranched or a branched alkene. The alkene can be, but is notlimited to, pentene, hexene, heptene, or octene.

In another aspect, the fermentation product is an amino acid. Theorganic acid can be, but is not limited to, aspartic acid, glutamicacid, glycine, lysine, serine, or threonine. See, for example, Richardand Margaritis, 2004, Biotechnology and Bioengineering 87(4): 501-515.

In another aspect, the fermentation product is a gas. The gas can be,but is not limited to, methane, H₂, CO₂, or CO. See, for example,Kataoka et al., 1997, Water Science and Technology 36(6-7): 41-47; andGunaseelan, 1997, Biomass and Bioenergy 13(1-2): 83-114.

In another aspect, the fermentation product is isoprene.

In another aspect, the fermentation product is a ketone. It will beunderstood that the term “ketone” encompasses a substance that containsone or more ketone moieties. The ketone can be, but is not limited to,acetone.

In another aspect, the fermentation product is an organic acid. Theorganic acid can be, but is not limited to, acetic acid, acetonic acid,adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid,formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronicacid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lacticacid, malic acid, malonic acid, oxalic acid, propionic acid, succinicacid, or xylonic acid. See, for example, Chen and Lee, 1997, Appl.Biochem. Biotechnol. 63-65: 435-448.

In another aspect, the fermentation product is polyketide.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Signal Peptides

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to17 of SEQ ID NO: 2 or amino acids 1 to 19 of SEQ ID NO: 4. Thepolynucleotide may further comprise a gene encoding a protein, which isoperably linked to the signal peptide. The protein is preferably foreignto the signal peptide. In one aspect, the polynucleotide encoding thesignal peptide is nucleotides 1 to 51 of SEQ ID NO: 1. In anotheraspect, the polynucleotide encoding the signal peptide is nucleotides 1to 57 of SEQ ID NO: 3.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides.

The present invention also relates to methods of producing a protein,comprising (a) cultivating a recombinant host cell comprising suchpolynucleotide; and optionally (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone, enzyme, receptor or portionthereof, antibody or portion thereof, or reporter. For example, theprotein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

Examples Strain

Corynascus thermophilus CBS 174.70 (synonym Myceliophthora fergusii;strain NN000308) was used as the source of the GH61 polypeptide codingsequences.

Media

PDA plates were composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

YPG medium was composed of 0.4% of yeast extract, 0.1% of KH₂PO₄, 0.05%of MgSO₄.7H₂O, and 1.5% glucose in deionized water.

YPM medium was composed of 1% yeast extract, 2% peptone, and 2% maltosein deionized water.

Minimal medium plates were composed of 342 g of sucrose, 20 ml of saltsolution, 20 g of agar, and deionized water to 1 liter.

Salt solution was composed of 2.6% KCl, 2.6% MgSO₄.7H₂O, 7.6% KH₂PO₄, 2ppm Na₂B₄O₇.10H₂O, 20 ppm CuSO₄.5H₂O, 40 ppm FeSO₄.7H₂O, 40 ppmMnSO₄.2H₂O, 40 ppm Na₂MoO₄.2H₂O, and 400 ppm ZnSO₄.7H₂O in deionizedwater.

Example 1: Corynascus thermophilus Genomic DNA Extraction

Corynascus thermophilus CBS 174.70 was inoculated onto a PDA plate andincubated for 3 days at 45° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 4 days at 45° C. with shaking at160 rpm. The mycelia were collected by filtration through MIRACLOTH®(Calbiochem, La Jolla, Calif., USA) and frozen in liquid nitrogen.Frozen mycelia were ground, by a mortar and a pestle, to a fine powder,and genomic DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGENGmbH, Hilden, Germany).

Example 2: Genome Sequencing, Assembly and Annotation

The extracted genomic DNA was delivered to the Beijing Genome Institute(BGI, Shenzhen, China) for genome sequencing using an ILLUMINA® GA2System (Illumina, Inc., San Diego, Calif., USA). The raw reads wereassembled at BGI using an in-house program SOAPdenovo (Li et al., 2010,Genome Research 20(2): 265-72). The assembled sequences were analyzedusing standard bioinformatics methods for gene finding and functionalprediction. Briefly, geneID (Parra et al., 2000, Genome Research 10(4):511-515) was used for gene prediction. Blastall version 2.2.10 (Altschulet al., 1990, J. Mol. Biol. 215(3): 403-410; National Center forBiotechnology Information (NCBI), Bethesda, Md., USA) and HMMER version2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda,Md., USA) were used to predict function based on structural homology.The family GH61 polypeptides were identified directly by analysis of theBlast results. The Agene program (Munch and Krogh, 2006, BMCBioinformatics 7: 263) and SignalP program (Nielsen et al., 1997,Protein Engineering 10: 1-6) were used to identify starting codons.SignalP was further used to predict the signal peptides. Pepstats (Riceet al., 2000, Trends Genet. 16(6): 276-277) was used to estimate theisoelectric points and molecular weights based on the deduced amino acidsequences.

Example 3: Cloning of Corynascus thermophilus GH61 Polypeptide Genesfrom Genomic DNA

Two GH61 polypeptide genes were selected for expression cloning as shownin Table 1.

TABLE 1 GH61 genes Gene name DNA sequence Protein sequence GH61_Mf2197SEQ ID NO: 1 SEQ ID NO: 2 GH61-Mf3680 SEQ ID NO: 3 SEQ ID NO: 4

Based on the DNA information obtained from genome sequencing,oligonucleotide primers, shown below, were designed to amplify thecoding sequences of the GH61 polypeptides from the genomic DNA ofCorynascus thermophilus CBS 174.70. The primers were synthesized byInvitrogen, Beijing, China.

SEQ ID NO: 1 forward primer: (SEQ ID NO: 5)ACACAACTGGGGATCCACCatgaagttcacctcgtccctcg SEQ ID NO: 1 reverse primer:(SEQ ID NO: 6) GTCACCCTCTAGATCTcgatcacgaaacccaccaagSEQ ID NO: 3 forward primer: (SEQ ID NO: 7)ACACAACTGGGGATCCACCatgaagggactcctcggcg SEQ ID NO: 3 reverse primer:(SEQ ID NO: 8) GTCACCCTCTAGATCTcgctgtcctcgtcctcctgLowercase characters of the forward primers represent the coding regionsof the gene and lowercase characters of the reverse primers representthe flanking region of the gene, while capitalized characters representregions homologous to insertion sites of pPFJO355 (WO 2011/005867).

For the GH61_Mf2197 gene, 20 picomoles of the SEQ ID NO: 1 forward andreverse primer pair were used in a PCR reaction composed of 2 μl ofCorynascus thermophilus genomic DNA, 10 μl of 5×GC Buffer (Finnzymes Oy,Espoo, Finland), 1.5 μl of DMSO, 2.5 mM each of dATP, dTTP, dGTP, anddCTP, and 0.6 unit of PHUSION™ High-Fidelity DNA Polymerase (FinnzymesOy, Espoo, Finland) in a final volume of 50 μl. The amplification wasperformed using a Peltier Thermal Cycler (MJ Research Inc., South SanFrancisco, Calif., USA) programmed for denaturing at 98° C. for 1minute; 5 cycles of denaturing each at 98° C. for 40 seconds, annealingat 65° C. for 40 seconds, with a 1° C. decrease per cycle and elongationat 72° C. for 150 seconds; 25 cycles each at 94° C. for 40 seconds, 60°C. for 40 seconds, and 72° C. for 150 seconds; and a final extension at72° C. for 5 minutes. The heat block then went to a 4° C. soak cycle.

For the GH61-Mf3680 gene, 20 picomoles of the SEQ ID NO: 3 forward andreverse primer pair were used in a PCR reaction composed of 2 μl ofCorynascus thermophilus genomic DNA, 5 μl of Pfu Buffer (FermentasInternational Inc., Burlington, Ontario, Canada), 2.5 mM each of dATP,dTTP, dGTP, and dCTP, and 0.6 unit of Pfu DNA Polymerase (FermentasInternational Inc., Burlington, Ontario, Canada) in a final volume of 50μl. The amplification was performed using a Peltier Thermal Cyclerprogrammed for denaturing at 94° C. for 3 minutes; 9 cycles ofdenaturing each at 94° C. for 40 seconds, annealing at 65° C. for 40seconds, with a 1° C. decrease per cycle and elongation at 72° C. for150 seconds; 25 cycles each at 94° C. for 40 seconds, 55° C. for 40seconds, and 72° C. for 150 seconds; and a final extension at 72° C. for10 minutes. The heat block then went to a 4° C. soak cycle.

The PCR products were isolated by 1.0% agarose gel electrophoresis using90 mM Tris-borate and 1 mM EDTA (TBE) buffer where a single product bandfrom each reaction (approximately 0.9 kb for GH61_Mf2197 andapproximately 1.4 kb for GH61-Mf3680) was visualized under UV light. ThePCR products were then purified from solution using an ILLUSTRA™ GFX™PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire,UK) according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Bam HI and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

An IN-FUSION® CF Dry-down Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) was used to clone the PCR fragments directlyinto the expression vector pPFJO355, without the need for restrictiondigestion and ligation.

TABLE 2 Gene name Plasmid DNA map GH61_Mf2197 pGH61_Mf2197 FIG. 1GH61-Mf3680 pGH61-Mf3680 FIG. 2

The PCR products and the digested vector were ligated together using theIN-FUSION® CF Dry-down Cloning Kit according to the manufacturer'sinstructions resulting in plasmids (Table 2) pGH61_Mf2197 (FIG. 1) andpGH61-Mf3680 (FIG. 2), in which transcription of each of the Corynascusthermophilus GH61 polypeptide coding sequences was under the control ofan Aspergillus oryzae alpha-amylase gene promoter. In brief, for eachligation reaction 30 ng of pPFJO355, digested with Bam HI and Bgl II,and 60 ng of the Corynascus thermophilus GH61 polypeptide purified PCRproduct were added to a reaction vial and resuspended in a final volumeof 10 μl by addition of deionized water. The reaction was incubated at37° C. for 15 minutes and then 50° C. for 15 minutes. Three μl of eachligation reaction were used to transform E. coli TOP10 competent cells(TIANGEN Biotech Co. Ltd., Beijing, China). E. coli transformantscontaining an expression construct were detected by colony PCR. ColonyPCR is a method for quick screening of plasmid inserts directly from E.coli colonies. Briefly, a single colony was transferred to a premixedPCR solution in a PCR tube, including PCR buffer, MgCl₂, dNTPs, andprimer pairs from which the PCR fragment was generated. Several colonieswere screened. After the PCR, reactions were analyzed by 1.0% agarosegel electrophoresis using TBE buffer. Plasmid DNA was prepared fromcolonies showing an insert with the expected size using a QIAPREP® SpinMiniprep Kit (QIAGEN GmbH, Hilden, Germany). The Corynascus thermophilusGH61 polypeptide coding sequences inserted in pGH61_Mf2197 andpGH61-Mf3680 were confirmed by DNA sequencing using 3730XL DNA Analyzers(Applied Biosystems Inc., Foster City, Calif., USA).

Example 4: Characterization of the Corynascus thermophilus Genomic DNASequences Encoding GH61 Polypeptides

The genomic DNA sequence and deduced amino acid sequence of theGH61_Mf2197 Corynascus thermophilus GH61 polypeptide gene are shown inSEQ ID NO: 1 and SEQ ID NO: 2, respectively. The coding sequence is 875bp including the stop codon, which is interrupted by 2 introns of 99 bp(nucleotides 56 to 154) and 77 bp (nucleotides 512 to 588). The encodedpredicted protein is 232 amino acids. Using the SignalP program (Nielsenet al., 1997, supra), a signal peptide of 17 residues was predicted. TheSignalP prediction is in accord with the necessity for having ahistidine reside at the N-terminus in order for proper metal binding andhence protein function to occur (See Harris et al., 2010, Biochemistry49: 3305, and Quinlan et al., 2011, Proc. Natl. Acad. Sci. USA 108:15079). The predicted mature protein contains 215 amino acids with apredicted molecular mass of 22.806 kDa and a predicted isoelectric pointof 6.38.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, supra) with a gap open penalty of 10, a gap extensionpenalty of 0.5, and the EBLOSUM62 matrix. The alignment showed that thededuced amino acid sequence of the GH61_Mf2197 Corynascus thermophilusgenomic DNA encoding a GH61 polypeptide shares 88.8% identity (excludinggaps) to the deduced amino acid sequence of a GH61 polypeptide fromMyceliophthora thermophila (UNIPROT G2QI82).

The genomic DNA sequence and deduced amino acid sequence of theGH61-Mf3680 Corynascus thermophilus GH61 polypeptide gene are shown inSEQ ID NO: 3 and SEQ ID NO: 4, respectively. The coding sequence is 1335bp including the stop codon, which is interrupted by 5 introns of 88 bp(nucleotides 285 to 372), 73 bp (nucleotides 447 to 519), 70 bp(nucleotides 562 to 631), 104 bp (nucleotides 847 to 950), and 121 bp(nucleotides 1118 to 1238). The encoded predicted protein is 292 aminoacids. Using the SignalP program (Nielsen et al., 1997, supra), a signalpeptide of 19 residues was predicted. The SignalP prediction is inaccord with the necessity for having a histidine reside at theN-terminus in order for proper metal binding and hence protein functionto occur (See Harris et al., 2010, supra, and Quinlan et al., 2011,supra). The predicted mature protein contains 273 amino acids with apredicted molecular mass of 28.73 kDa and a predicted isoelectric pointof 5.88.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, supra) with a gap open penalty of 10, a gap extensionpenalty of 0.5, and the EBLOSUM62 matrix. The alignment showed that thededuced amino acid sequence of the Corynascus thermophilus genomic DNAencoding a GH61 polypeptide shares 87.6% identity (excluding gaps) tothe deduced amino acid sequence of a GH61 polypeptide from Thielaviaheterothallica (UNIPROT G2Q4M0).

Example 5: Expression of the Corynascus thermophilus GH61 PolypeptideCoding Sequences in Aspergillus oryzae

Aspergillus oryzae HowB101 (WO 95/035385) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422 and transformed with 3 μg of pGH61_Mf2197 or pGH61-Mf3680.Each transformation yielded approximately 50 transformants. Eighttransformants from each transformation were isolated to individualMinimal medium plates.

Four transformants for each transformation were inoculated separatelyinto 3 ml of YPM medium in a 24-well plate and incubated at 30° C. withmixing at 150 rpm. After 3 days incubation, 20 μl of supernatant fromeach culture were analyzed by SDS-PAGE using a NUPAGE® NOVEX® 4-12%Bis-Tris Gel with 2-(N-morpholino)ethanesulfonic acid (MES) (InvitrogenCorporation, Carlsbad, Calif., USA) according to the manufacturer'sinstructions. The resulting gel was stained with INSTANTBLUE™ (ExpedeonLtd., Babraham Cambridge, UK). The SDS-PAGE profiles of the culturesshowed that the majority of the transformants had a major band at about25 kDa for GH61_Mf2197 and 40 kDa for GH61-Mf3680. The expressionstrains were designated A. oryzae O8M7W for GH61_Mf2197 and A. oryzaeO8KM3 for GH61-Mf3680.

Example 6: Fermentation of the Aspergillus oryzae Expression StrainsO8M7W and O8KM3

A slant was used to grow each expression strain (A. oryzae O8M7W and A.oryzae O8KM3) and then washed with 10 ml of YPM medium after 4 daysgrowth at 37° C. to fully grown spores. Each spore suspension wasinoculated into six 2-liter flasks, each containing 400 ml of YPMmedium, to generate broth for characterization of the GH61 polypeptides.The cultures were harvested on day 3 and filtered using a 0.45 μmDURAPORE® Membrane (Millipore, Bedford, Mass., USA).

Example 7: Purification of Recombinant Corynascus thermophilus GH61Polypeptides from Aspergillus oryzae 08M7W and 08KM3

A 2400 ml volume of the filtered broth of Aspergillus oryzae O8M7W wasprecipitated with ammonium sulfate (80% saturation) and re-dissolved in50 ml of 20 mM sodium acetate pH 5.5, dialyzed against the same buffer,and filtered through a 0.45 μm filter. The final volume was 115 ml. Thesolution was applied to a 40 ml Q SEPHAROSE® Fast Flow column (GEHealthcare, Piscataway, N.J., USA) equilibrated with 20 mM sodiumacetate pH 5.5. The proteins were eluted with a linear 0-0.25 M NaClgradient. Fractions were analyzed by SDS-PAGE using a NUPAGE® NOVEX®4-12% Bis-Tris Gel with MES. Fractions containing a band ofapproximately 25 kDa were pooled and the pooled solution wasconcentrated by ultrafiltration.

A 2400 ml volume of the filtered broth of Aspergillus oryzae 08KM3 wasprecipitated with ammonium sulfate (80% saturation) and re-dissolved in50 ml of 20 mM Tris-HCl pH 7.5, dialyzed against the same buffer, andfiltered through a 0.45 μm filter. The final volume was 75 ml. Thesolution was applied to a 40 ml Q SEPHAROSE® Fast Flow columnequilibrated with 20 mM Tris-HCl pH 7.5. The proteins were eluted with alinear 0-0.25 M NaCl gradient. Fractions were analyzed by SDS-PAGE usinga NUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES. Fractions containing aband of approximately 40 kDa were pooled and the pooled solution wasconcentrated by ultrafiltration.

Example 8: Pretreated Corn Stover Hydrolysis Assay

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 1.4 wt % sulfuric acid at 165°C. and 107 psi for 8 minutes. The water-insoluble solids in thepretreated corn stover (PCS) contained 56.5% cellulose, 4.6%hemicellulose and 28.4% lignin. Cellulose and hemicellulose weredetermined by a two-stage sulfuric acid hydrolysis with subsequentanalysis of sugars by high performance liquid chromatography using NRELStandard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid using NREL Standard Analytical Procedure#003.

Unmilled, unwashed PCS (whole slurry PCS) was prepared by adjusting thepH of the PCS to 5.0 by addition of 10 M NaOH with extensive mixing, andthen autoclaving for 20 minutes at 120° C. The dry weight of the wholeslurry PCS was 29%. Milled unwashed PCS (dry weight 32.35%) was preparedby milling whole slurry PCS in a Cosmos ICMG 40 wet multi-utilitygrinder (EssEmm Corporation, Tamil Nadu, India).

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates(Axygen, Union City, Calif., USA) in a total reaction volume of 1.0 ml.The hydrolysis was performed with 50 mg of insoluble PCS solids per mlof 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfateand various protein loadings of various enzyme compositions (expressedas mg protein per gram of cellulose). Enzyme compositions were preparedand then added simultaneously to all wells in a volume ranging from 50μl to 200 μl, for a final volume of 1 ml in each reaction. The plate wasthen sealed using an ALPS-300™ plate heat sealer, mixed thoroughly, andincubated at a specific temperature for 72 hours. All experimentsreported were performed in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate and the filtrates were analyzed for sugar contentas described below. When not used immediately, filtered aliquots werefrozen at −20° C. The glucose concentration of the samples diluted in0.005 M H₂SO₄ was measured using a 4.6×250 mm AMINEX® HPX-87H column byelution with 0.05% w/w benzoic acid-0.005 M H₂SO₄ at 65° C. at a flowrate of 0.6 ml per minute, and quantitation by integration of theglucose signal from refractive index detection (CHEMSTATION®, AGILENT®1100 HPLC) calibrated by a pure glucose standard. The resultant glucoseequivalents were used to calculate the percentage of celluloseconversion for each reaction.

The measured glucose concentrations were adjusted for the appropriatedilution factor. The net concentrations of enzymatically-producedglucose from the milled unwashed PCS were determined by adjusting themeasured glucose concentrations for corresponding background glucoseconcentrations in the milled unwashed PCS at a zero time point. All HPLCdata processing was performed using MICROSOFT EXCEL™ software.

The degree of cellulose conversion to glucose was calculated using thefollowing equation: % conversion=(glucose concentration/glucoseconcentration in a limit digest)×100. In order to calculate %conversion, a 100% conversion point was set based on a cellulase control(100 mg of Trichoderma reesei cellulase per gram cellulose), and allvalues were divided by this number and then multiplied by 100.Triplicate data points were averaged and standard deviation wascalculated.

Example 9: Preparation of an Enzyme Composition

The Aspergillus fumigatus GH7A cellobiohydrolase I (GENESEQP:AZH96970)was prepared recombinantly in Aspergillus oryzae as described in WO2011/057140. The filtered broth of the A. fumigatus cellobiohydrolase Iwas concentrated and buffer exchanged using a tangential flowconcentrator (Pall Filtron, Northborough, Mass., USA) equipped with a 10kDa polyethersulfone membrane (Pall Filtron, Northborough, Mass., USA)with 20 mM Tris-HCl pH 8.0. The desalted broth of the A. fumigatuscellobiohydrolase I was loaded onto a Q SEPHAROSE® ion exchange column(GE Healthcare, Piscataway, N.J., USA) equilibrated in 20 mM Tris-HCl pH8 and eluted using a linear 0 to 1 M NaCl gradient. Fractions werecollected and fractions containing the cellobiohydrolase I were pooledbased on SDS-PAGE analysis using 8-16% CRITERION® Stain-free SDS-PAGEgels (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

The Aspergillus fumigatus GH6A cellobiohydrolase II (GENESEQP:AZH96972)was prepared recombinantly in Aspergillus oryzae as described in WO2011/057140. The filtered broth of the A. fumigatus cellobiohydrolase IIwas buffer exchanged into 20 mM Tris pH 8.0 using a 400 ml SEPHADEX™G-25 column (GE Healthcare, United Kingdom). The fractions were pooledand adjusted to 1.2 M ammonium sulphate-20 mM Tris pH 8.0. Theequilibrated protein was loaded onto a PHENYL SEPHAROSE™ 6 Fast Flowcolumn (high sub) (GE Healthcare, Piscataway, N.J., USA) equilibrated in20 mM Tris pH 8.0 with 1.2 M ammonium sulphate, and bound proteins wereeluted with 20 mM Tris pH 8.0 with no ammonium sulphate. The fractionswere pooled.

The Trichoderma reesei GH5 endoglucanase II (GENESEQP:BAM80436) wasprepared recombinantly according to WO 2011/057140 using Aspergillusoryzae as a host. The filtered broth of the T. reesei endoglucanase IIwas desalted and buffer-exchanged into 20 mM Tris pH 8.0 using atangential flow concentrator equipped with a 10 kDa polyethersulfonemembrane.

The Aspergillus fumigatus GH10 xylanase (xyn3) (GENESEQP:AEC74753) wasprepared recombinantly according to WO 2006/078256 using Aspergillusoryzae BECh2 (WO 2000/39322) as a host. The filtered broth of the A.fumigatus xylanase was desalted and buffer-exchanged into 50 mM sodiumacetate pH 5.0 using a HIPREP® 26/10 Desalting Column (GE Healthcare,Piscataway, N.J., USA).

The Aspergillus fumigatus Cel3A beta-glucosidase (GENESEQP:AEA33207) wasrecombinantly prepared according to WO 2005/047499 using Aspergillusoryzae as a host. The filtered broth of Aspergillus fumigatus Cel3Abeta-glucosidase was concentrated and buffer exchanged using atangential flow concentrator equipped with a 10 kDa polyethersulfonemembrane with 20 mM Tris-HCl pH 8.5. The sample was loaded onto a QSEPHAROSE® High Performance column (GE Healthcare, Piscataway, N.J.,USA) equilibrated in 20 mM Tris pH 8.5, and bound proteins were elutedwith a linear gradient from 0-600 mM sodium chloride. Fractions werecollected and fractions containing the Aspergillus fumigatus Cel3Abeta-glucosidase were pooled based on SDS-PAGE analysis using 8-16%CRITERION® Stain-free SDS-PAGE gels (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA). The fractions were concentrated and loaded ontoa SUPERDEX® 75 HR 26/60 column equilibrated with 20 mM Tris-150 mMsodium chloride pH 8.5. Fractions were collected and fractionscontaining the Aspergillus fumigatus Cel3A beta-glucosidase were pooledbased on SDS-PAGE analysis using 8-16% CRITERION® Stain-free SDS-PAGEgels.

The Aspergillus fumigatus NN051616 GH3 beta-xylosidase(GENESEQP:AZI05042) was prepared recombinantly in Aspergillus oryzae asdescribed in WO 2011/057140. The filtered broth of the A. fumigatusbeta-xylosidase was desalted and buffer-exchanged into 50 mM sodiumacetate pH 5.0 using a HIPREP® 26/10 Desalting Column.

The protein concentration for each of the monocomponents described abovewas determined using a Microplate BCA™ Protein Assay Kit (Thermo FischerScientific, Waltham, Mass., USA) in which bovine serum albumin was usedas a protein standard. An enzyme composition was prepared composed ofeach monocomponent as follows: 37% Aspergillus fumigatus Cel7Acellobiohydrolase I, 25% Aspergillus fumigatus Cel6A cellobiohydrolaseII, 10% Trichoderma reesei GH5 endoglucanase II, 5% Aspergillusfumigatus GH10 xylanase, 5% Aspergillus fumigatus beta-glucosidase, and3% Aspergillus fumigatus beta-xylosidase. The enzyme composition isdesignated herein as “cellulolytic enzyme composition”.

Example 10: Effect of the GH61_Mf2197 Corynascus thermophilus GH61Polypeptide on the Hydrolysis of Milled Unwashed PCS by a CellulolyticEnzyme Composition

The GH61_Mf2197 Corynascus thermophilus GH61 polypeptide (SEQ ID NO: 2)was evaluated for the ability to enhance the hydrolysis of milledunwashed PCS (Example 8) by the cellulolytic enzyme composition ofExample 9 at 2.55 mg total protein per g cellulose at 50° C. The C.thermophilus GH61 polypeptide was added at 0.45 mg protein per gcellulose.

The assay was performed as described in Example 8. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in Table I, the cellulolytic enzyme composition that includedthe C. thermophilus GH61 polypeptide outperformed the cellulolyticenzyme composition (2.55 mg protein per g cellulose) without GH61polypeptide at 50° C. The degree of cellulose conversion to glucose forthe C. thermophilus GH61 polypeptide added to the cellulolytic enzymecomposition was higher than the cellulolytic enzyme composition withoutadded GH61 at 50° C.

TABLE I % Cellulose to Glucose Standard Conversion DeviationCellulolytic Enzyme Composition 50° C. 50° C. Cellulolytic EnzymeComposition 40.58 0.32 no GH61 (2.55 mg/g) Cellulolytic EnzymeComposition 46.84 0.36 (2.55 mg/g) with Corynascus thermophilus GH61(0.45 mg/g)

Example 11: Effect of the GH61-Mf3680 Corynascus thermophilus GH61Polypeptide on the Hydrolysis of Milled Unwashed PCS by a CellulolyticEnzyme Composition

The GH61-Mf3680 Corynascus thermophilus GH61 polypeptide (SEQ ID NO: 4)was evaluated for the ability to enhance the hydrolysis of milledunwashed PCS (Example 8) by the cellulolytic enzyme composition ofExample 9 at 2.55 mg total protein per g cellulose at 50° C., 55° C.,and 60° C. The C. thermophilus GH61 polypeptide was added at 0.45 mgprotein per g cellulose.

The assay was performed as described in Example 8. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in Table II, the cellulolytic enzyme composition that includedthe C. thermophilus GH61 polypeptide outperformed the cellulolyticenzyme composition (2.55 mg protein per g cellulose) without GH61polypeptide at 50° C., 55° C., and 60° C. The degree of celluloseconversion to glucose for the C. thermophilus GH61 polypeptide added tothe cellulolytic enzyme composition was higher than the cellulolyticenzyme composition without added GH61 at 50° C., 55° C., and 60° C.

TABLE II % Cellulose to Glucose Conversion Standard DeviationCellulolytic Enzyme Composition 50° C. 55° C. 60° C. 50° C. 55° C. 60°C. Cellulolytic Enzyme Composition no 40.58 48.90 43.76 0.32 0.70 0.42GH61 (2.55 mg/g) Cellulolytic Enzyme Composition 44.86 51.56 46.51 1.480.41 0.36 (2.55 mg/g) with Corynascus thermophilus GH61 (0.45 mg/g)

The present invention is further described by the following numberedparagraphs:

[1] An isolated polypeptide having cellulolytic enhancing activity,selected from the group consisting of: (a) a polypeptide having at least90% sequence identity to the mature polypeptide of SEQ ID NO: 2 or SEQID NO: 4; (b) a polypeptide encoded by a polynucleotide that hybridizesunder at least very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3; the cDNAsequence thereof; or the full-length complement thereof; (c) apolypeptide encoded by a polynucleotide having at least 90% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 orSEQ ID NO: 3; or the cDNA sequence thereof; (d) a variant of the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 comprising a substitution,deletion, and/or insertion at one or more positions; and (e) a fragmentof the polypeptide of (a), (b), (c), or (d) that has cellulolyticenhancing activity.

[2] The polypeptide of paragraph 1, having at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to themature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

[3] The polypeptide of paragraph 1 or 2, which is encoded by apolynucleotide that hybridizes under very high stringency conditionswith the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ IDNO: 3; the cDNA sequence thereof; or the full-length complement thereof.

[4] The polypeptide of any of paragraphs 1-3, which is encoded by apolynucleotide having at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or the cDNA sequencethereof.

[5] The polypeptide of any of paragraphs 1-4, comprising or consistingof SEQ ID NO: 2 or SEQ ID NO: 4 or the mature polypeptide of SEQ ID NO:2 or the mature polypeptide of SEQ ID NO: 4.

[6] The polypeptide of paragraph 5, wherein the mature polypeptide isamino acids 18 to 232 of SEQ ID NO: 2 or amino acids 20 to 292 of SEQ IDNO: 4.

[7] The polypeptide of any of paragraphs 1-4, which is a variant of themature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 comprising asubstitution, deletion, and/or insertion at one or more positions.

[8] The polypeptide of paragraph 1, which is a fragment of SEQ ID NO: 2or SEQ ID NO: 4, wherein the fragment has cellulolytic enhancingactivity.

[9] The polypeptide of any of paragraphs 1-8, which is encoded by thepolynucleotide contained in Corynascus thermophilus CBS 174.70.

[10] An isolated polypeptide comprising a catalytic domain selected fromthe group consisting of: (a) a catalytic domain having at least 90%sequence identity to amino acids 20 to 223 of SEQ ID NO: 4; (b) acatalytic domain encoded by a polynucleotide that hybridizes under atleast very high stringency conditions with nucleotides 58 to 1004 of SEQID NO: 3; the cDNA sequence thereof; or the full-length complementthereof; (c) a catalytic domain encoded by a polynucleotide having atleast 90% sequence identity to nucleotides 58 to 1004 of SEQ ID NO: 3 orthe cDNA sequence thereof; (d) a variant of amino acids 20 to 223 of SEQID NO: 4 comprising a substitution, deletion, and/or insertion at one ormore positions; and (e) a fragment of the catalytic domain of (a), (b),(c), or (d) that has cellulolytic enhancing activity.

[11] The polypeptide of paragraph 10, further comprising a carbohydratebinding module.

[12] An isolated polypeptide comprising a carbohydrate binding moduleoperably linked to a catalytic domain, wherein the binding domain isselected from the group consisting of: (a) a carbohydrate binding modulehaving at least 90% sequence identity to amino acids 255 to 292 of SEQID NO: 4; (b) a carbohydrate binding module encoded by a polynucleotidethat hybridizes under at least very high stringency conditions withnucleotides 1098 to 1332 of SEQ ID NO: 3; the cDNA sequence thereof; orthe full-length complement thereof; (c) a carbohydrate binding moduleencoded by a polynucleotide having at least 90% sequence identity tonucleotides 1098 to 1332 of SEQ ID NO: 3 or the cDNA sequence thereof;(d) a variant of amino acids 255 to 292 of SEQ ID NO: 4 comprising asubstitution, deletion, and/or insertion at one or more positions; and(e) a fragment of the carbohydrate binding module of (a), (b), (c), or(d) that has binding activity.

[13] The polypeptide of paragraph 12, wherein the catalytic domain isobtained from a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, xylanase, or beta-xylosidase.

[14] A composition comprising the polypeptide of any of paragraphs 1-13.

[15] An isolated polynucleotide encoding the polypeptide of any ofparagraphs 1-13.

[16] A nucleic acid construct or expression vector comprising thepolynucleotide of paragraph 15 operably linked to one or more controlsequences that direct the production of the polypeptide in an expressionhost.

[17] A recombinant host cell comprising the polynucleotide of paragraph15 operably linked to one or more control sequences that direct theproduction of the polypeptide.

[18] A method of producing the polypeptide of any of paragraphs 1-13,comprising: cultivating a cell, which in its wild-type form produces thepolypeptide, under conditions conducive for production of thepolypeptide.

[19] The method of paragraph 18, further comprising recovering thepolypeptide.

[20] A method of producing a polypeptide having cellulolytic enhancingactivity, comprising: cultivating the host cell of paragraph 17 underconditions conducive for production of the polypeptide.

[21] The method of paragraph 20, further comprising recovering thepolypeptide.

[22] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs 1-13.

[23] A method of producing a polypeptide having cellulolytic enhancingactivity, comprising: cultivating the transgenic plant or plant cell ofparagraph 22 under conditions conducive for production of thepolypeptide.

[24] The method of paragraph 23, further comprising recovering thepolypeptide.

[25] A method of producing a mutant of a parent cell, comprisinginactivating a polynucleotide encoding the polypeptide of any ofparagraphs 1-13, which results in the mutant producing less of thepolypeptide than the parent cell.

[26] A mutant cell produced by the method of paragraph 25.

[27] The mutant cell of paragraph 26, further comprising a gene encodinga native or heterologous protein.

[28] A method of producing a protein, comprising: cultivating the mutantcell of paragraph 26 or 27 under conditions conducive for production ofthe protein.

[29] The method of paragraph 28, further comprising recovering theprotein.

[30] A double-stranded inhibitory RNA (dsRNA) molecule comprising asubsequence of the polynucleotide of paragraph 15, wherein optionallythe dsRNA is a siRNA or a miRNA molecule.

[31] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph30, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or moreduplex nucleotides in length.

[32] A method of inhibiting the expression of a polypeptide havingcellulolytic enhancing activity in a cell, comprising administering tothe cell or expressing in the cell the double-stranded inhibitory RNA(dsRNA) molecule of paragraph 30 or 31.

[33] A cell produced by the method of paragraph 32.

[34] The cell of paragraph 33, further comprising a gene encoding anative or heterologous protein.

[35] A method of producing a protein, comprising: cultivating the cellof paragraph 34 or 34 under conditions conducive for production of theprotein.

[36] The method of paragraph 35, further comprising recovering theprotein.

[37] An isolated polynucleotide encoding a signal peptide comprising orconsisting of amino acids 1 to 17 of SEQ ID NO: 2 or amino acids 1 to 19of SEQ ID NO: 4.

[38] A nucleic acid construct or expression vector comprising a geneencoding a protein operably linked to the polynucleotide of paragraph37, wherein the gene is foreign to the polynucleotide encoding thesignal peptide.

[39] A recombinant host cell comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph 37, wherein the geneis foreign to the polynucleotide encoding the signal peptide.

[40] A method of producing a protein, comprising: cultivating arecombinant host cell comprising a gene encoding a protein operablylinked to the polynucleotide of paragraph 37, wherein the gene isforeign to the polynucleotide encoding the signal peptide, underconditions conducive for production of the protein.

[41] The method of paragraph 40, further comprising recovering theprotein.

[42] A process for degrading a cellulosic material, comprising: treatingthe cellulosic material with an enzyme composition in the presence ofthe polypeptide having cellulolytic enhancing activity of any ofparagraphs 1-13.

[43] The process of paragraph 42, wherein the cellulosic material ispretreated.

[44] The process of paragraph 42 or 43, wherein the enzyme compositioncomprises one or more enzymes selected from the group consisting of acellulase, a polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin.

[45] The process of paragraph 44, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[46] The process of paragraph 44, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[47] The process of any of paragraphs 42-46, further comprisingrecovering the degraded cellulosic material.

[48] The process of paragraph 47, wherein the degraded cellulosicmaterial is a sugar.

[49] The process of paragraph 48, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[50] A process for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with an enzyme composition in thepresence of the polypeptide having cellulolytic enhancing activity ofany of paragraphs 1-13; (b) fermenting the saccharified cellulosicmaterial with one or more fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.

[51] The process of paragraph 50, wherein the cellulosic material ispretreated.

[52] The process of paragraph 50 or 51, wherein the enzyme compositioncomprises the enzyme composition comprises one or more enzymes selectedfrom the group consisting of a cellulase, a polypeptide havingcellulolytic enhancing activity, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

[53] The process of paragraph 52, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[54] The process of paragraph 52, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[55] The process of any of paragraphs 50-54, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[56] The process of any of paragraphs 50-55, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[57] A process of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more fermentingmicroorganisms, wherein the cellulosic material is saccharified with anenzyme composition in the presence of the polypeptide havingcellulolytic enhancing activity of any of paragraphs 1-13.

[58] The process of paragraph 57, wherein the fermenting of thecellulosic material produces a fermentation product.

[59] The process of paragraph 58, further comprising recovering thefermentation product from the fermentation.

[60] The process of any of paragraphs 57-59, wherein the cellulosicmaterial is pretreated before saccharification.

[61] The process of any of paragraphs 57-60, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a polypeptide having cellulolytic enhancingactivity, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[62] The process of paragraph 61, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[63] The process of paragraph 61, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[64] The process of any of paragraphs 58-63, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[65] A whole broth formulation or cell culture composition comprisingthe polypeptide of any of paragraphs 1-13.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1-20. (canceled)
 21. A nucleic acid construct comprising apolynucleotide encoding a GH61 polypeptide having cellulolytic enhancingactivity, wherein the polynucleotide is operably linked to one or moreheterologous control sequences that direct the production of the GH61polypeptide in a recombinant host cell, and wherein the GH61 polypeptidehaving cellulolytic enhancing is selected from the group consisting of:(a) a GH61 polypeptide having at least 95% sequence identity to themature polypeptide of h SEQ ID NO: 4; (b) a GH61 polypeptide encoded bya polynucleotide that hybridizes under very high stringency conditionswith the full-length complement of the mature polypeptide codingsequence of SEQ ID NO: 3 or the cDNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 3; and (c) a GH61 polypeptide encoded by apolynucleotide having at least 98% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 or the cDNA sequence of themature polypeptide coding sequence of SEQ ID NO:
 3. 22. The nucleic acidconstruct of claim 21, wherein the GH61 polypeptide has at least 95%sequence identity to the mature polypeptide of SEQ ID NO:
 4. 23. Thenucleic acid construct of claim 21, wherein the GH61 polypeptide has atleast 96% sequence identity to the mature polypeptide of SEQ ID NO: 4.24. The nucleic acid construct of claim 21, wherein the GH61 polypeptidehas at least 97% sequence identity to the mature polypeptide of SEQ IDNO:
 4. 25. The nucleic acid construct of claim 21, wherein the GH61polypeptide has at least 98% sequence identity to the mature polypeptideof SEQ ID NO:
 4. 26. The nucleic acid construct of claim 21, wherein theGH61 polypeptide has at least 99% sequence identity to the maturepolypeptide of SEQ ID NO:
 4. 27. The nucleic acid construct of claim 21,wherein the GH61 polypeptide comprises SEQ ID NO: 4 or the maturepolypeptide of SEQ ID NO:
 4. 28. The nucleic acid construct of claim 21,wherein the GH61 polypeptide is a fragment of the mature polypeptide ofSEQ ID NO: 4, and wherein the fragment has cellulolytic enhancingactivity.
 29. A recombinant host cell transformed with the nucleic acidconstruct of claim
 21. 30. A method of producing a GH61 polypeptidehaving cellulolytic enhancing activity, comprising: cultivating therecombinant host cell of claim 29 under conditions conducive forproduction of the GH61 polypeptide.
 31. A transgenic plant, plant partor plant cell transformed with the nucleic acid construct of claim 21.32. A method of producing a GH61 polypeptide having cellulolyticenhancing activity, comprising: cultivating the transgenic plant orplant cell of claim 31 under conditions conducive for production of theGH61 polypeptide, and recovering the GH61 polypeptide.
 33. A recombinanthost cell transformed with a nucleic acid construct comprising apolynucleotide encoding a GH61 polypeptide having cellulolytic enhancingactivity, wherein the polynucleotide is operably linked to one or morecontrol sequences that direct the production of the GH61 polypeptide inthe recombinant host cell, wherein the GH61 polypeptide is heterologousto the recombinant host cell, and wherein the GH61 polypeptide havingcellulolytic enhancing is selected from the group consisting of: (a) aGH61 polypeptide having at least 95% sequence identity to the maturepolypeptide of h SEQ ID NO: 4; (b) a GH61 polypeptide encoded by apolynucleotide that hybridizes under very high stringency conditionswith the full-length complement of the mature polypeptide codingsequence of SEQ ID NO: 3 or the cDNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 3; and (c) a GH61 polypeptide encoded by apolynucleotide having at least 98% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 or the cDNA sequence of themature polypeptide coding sequence of SEQ ID NO:
 3. 34. The recombinanthost cell of claim 33, wherein the GH61 polypeptide has at least 95%sequence identity to the mature polypeptide of SEQ ID NO:
 4. 35. Therecombinant host cell of claim 33, wherein the GH61 polypeptide has atleast 96% sequence identity to the mature polypeptide of SEQ ID NO: 4.36. The recombinant host cell of claim 33, wherein the GH61 polypeptidehas at least 97% sequence identity to the mature polypeptide of SEQ IDNO:
 4. 37. The recombinant host cell of claim 33, wherein the GH61polypeptide has at least 98% sequence identity to the mature polypeptideof SEQ ID NO:
 4. 38. The recombinant host cell of claim 33, wherein theGH61 polypeptide has at least 99% sequence identity to the maturepolypeptide of SEQ ID NO:
 4. 39. The recombinant host cell of claim 33,wherein the GH61 polypeptide comprises SEQ ID NO: 4 or the maturepolypeptide of SEQ ID NO:
 4. 40. The recombinant host cell of claim 33,wherein the GH61 polypeptide is a fragment of the mature polypeptide ofSEQ ID NO: 4, and wherein the fragment has cellulolytic enhancingactivity.
 41. A method of producing a GH61 polypeptide havingcellulolytic enhancing activity, comprising: cultivating the recombinanthost cell of claim 33 under conditions conducive for production of theGH61 polypeptide, and recovering the GH61 polypeptide.